“All things are poison and nothing is without poison, only the dose permits something not to be poisonous”
-Paracelsus (1493 –1541)
Toxicology is a branch of science that deals with detection, occurrence, properties, effects and regulation of any substance, which tend to produce harmful effects to living organisms.
Toxicological studies were performed not only to ensure the safety but also as a part of developing selective toxicants, such as anticancer drugs, other clinical drugs and pesticides. Toxicity is otherwise a quantitative as well as a qualitative phenomenon.There exists a limit between harmless to toxic effects, for example metals such as iron, copper, cobalt, magnesium and zinc are essential in our diet at low concentration but leads to toxicity when it exceeds a certain dose. Thus dose response relationship also found to have direct relationship with the toxicity.
Measuring toxicity is complex as it may vary from organ to organ, age, biological conditions, genetics, diet etc. Carbon tetrachloride is harmless to chicken whereas it act as a potent hepatotoxic in many other species. Compounds may have toxic effects in certain conditions but not others, compounds may not have toxic effect when combined with other compounds. Route of exposure to a toxicant is also an important factor to be considered as toxic effect from intravenous (IV), intraperitoneal (IP), intramuscular (IM), or subcutaneous injection (SC) of a same compound will be quite different (Ernest Hodgson (2004)).
Phillipus Aureoles born in 1493 in Switzerland. He took pseudonym of Theophrastus Bombastus von Hohenheim, later known as Paracelsus and he is considered as the father of toxicology. He stated that “All substances are poisons; there is none, which is not a poison. The right dose differentiates a poison from a remedy.
Mathieu Joseph Bonaventure Orfila (1787-1853), a French toxicologist and chemist known as father of modern toxicology because of his work in analytical toxicology related to arsenic.
The Ebrus papyrus, an ancient Egyptian record dated approximately 1500BC contains a wider range of toxic substance including hemlock and aconite. She Nung, father of Chinese medicine (approximately 2695 BCE), who was noted for 365 herbs and dying from a toxic overdose, also wrote an early treatise, On herbal medical experiments Poisons modified through ages and ultimately helped China to become the leader in herbal medicine.
German military in World War I released chlorine gas over the battlefield at Ypres salient in Belgium lead to death of estimated 5000 French and Algerian troops. In World War II there was the use of various powerful nerve gases.In 1984 release of Methyl Isocynate by a union carbide subsidiary manufacturing pesticides in Bhopal, India resulted in the death of thousands and injury of hundreds of thousands. A strain of anthrax, the bacterium bacillus anthracis killed several people in US in 1991. In 2006 Alexander Livienko, who was poisoned by exposure to the radioactive polonium 210.
Two Basic Functions of Toxicology
1.Assess the likelihood of the occurrence of adverse effects (qualitative)
Is It Safe?
2.Study the nature and mode of action of adverse effects (quantitative)
At what concentration is it safe?
Dose Response Assessment
No Observed Adverse Effect Level (NOAEL): The highest exposure level at which there are no biologically significant increases in the frequency or severity of adverse effect between the exposed population and its appropriate control; some effects may be produced at this level, but they are not considered adverse effects.
Lowest Observed Adverse Effect Level (LOAEL): The lowest exposure level at which there are biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control group.
The units of NOAEL or LOAEL: mg/kg/bw/day or ppm. For inhalation route, the unit can be mg/L/6h/day.
NOAEL is not only used for determining the hazard classification of the specific target organ systematic toxicity of a chemical substance, but also used to derive threshold safety exposure dose to humans such as derived no effect level (DNEL), occupational exposure limit (OEL) and acceptable daily intake (ADI).
Biologically Significant Effect
A response (to a stimulus) in an organism or other biological system that is considered to have substantial or noteworthy effect (positive or negative) on the well-being of the biological system. The concept is to be distinguished from statistically significant effects or changes, which may or may not be meaningful to the general state of health of the system.
A change in morphology, physiology, growth, development, reproduction, or life span of a cell or organism, system, or (sub)population that results in an impairment of functional capacity, an impairment of the capacity to compensate for additional stress, or an increase in susceptibility to other influences.
The process whereby a cell or organism responds to a xenobiotic so that the cell or organism will survive in the new environment that contains the xenobiotic without impairment of function.
Histological changes in the respiratory tract which may be assessed as adaptive responses:
Mucous cell hyperplasia may be induced by dehydration of the nasal epithelium or inhalation of
aerosols of a variety of chemicals.
Exposure to aerosols may cause transitional epithelium at the base of the epiglottis in rats to
change into squamous epithelium.
Macrophage accumulation in the lung after exposure to low solubility materials in the absence
of any signs of inflammatory reaction is considered to be a physiological sign of enhanced
alveolar clearance activity.
In order to further enhance alveolar clearance, alveolar epithelium of the lung may be replaced
by ciliated epithelium. This process is called bronchiolization and represents the ultimate
adaptive response after exposures to high concentrations of chemical
The Threshold of Toxicological Concern (TTC)
The Threshold of Toxicological Concern (TTC) is a science-based pragmatic tool for prioritizing those chemicals with low-level exposures that require more data over those that can be presumed to present no appreciable human health risk
A benchmark dose (BMD) is a dose or concentration that produces a predetermined change in the response rate of an adverse effect. This predetermined change in response is called the benchmark response (BMR).
Normally, the default BMR is 5% or 10% change in the response rate of an adverse effect relative to the response of control group depending on whether response data is continuous or quantal(dichotomous).
The values of a biological parameter in individual animals
Count of red blood cells
The fraction of animals with a specific effect in a population
Tumour incidence rate
Percentage of animals developing an adverse effect
Margin of Safety
The Margin of Safety ( MOS ) is usually calculated as the ratio of the toxic dose to 1% of the population (TD01) to the dose that is 99% effective to the population ( ED99 )
The Therapeutic Index (TI) is used to compare the therapeutically effective dose to the toxic dose of a pharmaceutical agent. The TI is a statement of relative safety of a drug. It is the ratio of the dose that produces toxicity to the dose needed to produce the desired therapeutic response. The common method used to derive the TI is to use the 50% dose-response points, including TD50 (toxic dose) and ED50 (effective dose).
Therapeutic Index = Toxic dose/Dose for therapeutic response = TD50/ED50
Example: if the TD50 is 200 and the ED50 is 20 mg, the TI would be 10 mg.
(More TI less safer and more toxic the drug would be)
Weight of Evidence (WoE)
The weight of evidence approach means that you use a combination of information from several independent sources to give sufficient evidence to fulfil an information requirement.
High dose selection criteria in a toxicology study (In vivo)
There are five general criteria for defining the high dose in a toxicology study.
(i) Maximum tolerated dose
(ii) Limit dose
(iii) Top dose based on saturation of exposure
(iv) Maximum feasible/practical dose, or
(v) Dose providing a 50-fold margin of exposure.
Human Equivalent Animal Dose Calculation
Animal dose(mg/kg) =Human dose (mg/kg) x Conversion Factor
Default Human Body Weight - 60 kg
Eg. if Human dose - 120 mg/day, Human equivalent rat dose?
Human equivalent rat dose(mg/kg) = (120/60(human bwt)) x 6.17
ie. =12.34 mg/kg
Algae Growth Inhibition Test
Acute Immobilization Test in Daphnia
Daphnia magna Reproduction Test
Acute Fish Toxicity Test
Lemna Growth Inhibition Test
Honeybee – Acute Oral and Contact Toxicity Test
Earthworm – Acute Toxicity Test
Earthworm – Reproduction Toxicity Test
Silkworm acute and chronic Toxicity tests
Avian Acute Oral Toxicity Test
Avian Dietary Toxicity Test
Ames test- Reverse mutation assay
Chromosomal Aberration Assay in Human Lymphocytes CHO Cell Line
Mouse Lymphoma Assay in L5178Y Ttk+/- Cells
In vitro Micronucleus Assay in Human Lymphocytes
Sister Chromatid Exchange Assay
Other Toxicology Studies
Dermal Irritation and Eye Irritation
Repeated Dose Toxicity Studies
Developmental & Reproductive Toxicology
Fertility and Early Embryonic Development to Implantation Study (Segment I)
Pre-and Post-natal Development Study (Segment III)
Embryo-Fetal Development Study (Segment II)
List of ICH Safety Guidelines
S1A - S1C Carcinogenicity Studies
S2 Genotoxicity Studies
S3A - S3B Toxicokinetics and Pharmacokinetics
S4 Toxicity Testing
S5 Reproductive Toxicology
S6 Biotechnological Products
S7A - S7B Pharmacology Studies
S8 Immunotoxicology Studies
S9 Nonclinical Evaluation for Anticancer Pharmaceuticals
S10 Photosafety Evaluation
S11 Nonclinical Paediatric Safety
Definition of Risk Assessment
Risk assessment is the systematic scientiﬁc evaluation of potential adverse health effects resulting from human exposures to hazardous agents or situations.
Steps Involved in Risk Assessment
Step 1 - Hazard Identification
Examines whether a stressor has the potential to cause harm to humans
and/or ecological systems, and if so, under what circumstances.
Step 2 - Dose-Response Assessment
Examines the numerical relationship between exposure and effects.
Step 3 - Exposure Assessment
Examines what is known about the frequency, timing, and levels of contact
with a stressor.
Step 4 - Risk Characterization
Examines how well the data support conclusions about the nature and
extent of the risk from exposure to environmental stressors.
PDE: Permitted Daily Exposure (ADE Allowable Daily Exposure)
OEL occupational exposure limit
OEV occupational exposure values
OEB occupational exposure bands
Permitted Daily Exposure (ADE Allowable Daily Exposure)
A substance specific dose that is unlikely to cause an adverse effect if an individual is exposed at or below this dose every day for a lifetime
Formula for calculation of PDE
PDE = (NOAEL x Weight Adjustment) / (F1 x F2 x F3 x F4 x F5)
F1: A factor (values between 2 and 12) to account for extrapolation between
Adjustment Factor F1 Species wise
Human - 1
Dogs - 2
Rabbits - 2.5
Rats - 5
Mouse - 12
Other 10 (or calculated)
F2: A factor of 10 to account for variability between individuals
Adjustment factor F2 – Variability amongst individuals
• Typically defaulted to 10
F3: A factor 10 to account for repeat-dose toxicity studies of short duration, i.e.,
less than 4-weeks
F4: A factor (1-10) that may be applied in cases of severe toxicity, e.g. non-
genotoxic carcinogenicity, neurotoxicity or teratogenicity
1 for fetal toxicity associated with maternal toxicity
5 for fetal toxicity without maternal toxicity
5 for a teratogenic effect with maternal toxicity
10 for a teratogenic effect without maternal toxicity
F5: A variable factor that may be applied if the no-effect level was not
established. When only an LOEL is available, a factor of up to 10 could be
used depending on the severity of the toxicity.
Point of Departure
In toxicology, point of departure (POD) is defined as the point on a toxicological dose-response curve established from experimental data or observational data generally corresponding to an estimated low effect level or no effect level. It marks the beginning of extrapolation to toxicological reference dose RfD or reference concentration RfC.
Occupational Exposure Limit (OEL)
An occupational exposure limit is an upper limit on the acceptable concentration of a hazardous substance in workplace air for a particular material or class of materials. It is typically set by competent national authorities and enforced by legislation to protect occupational safety and health
Calculation of an OEL for systemic effects
OEL (mg/m3) = (POD x BW) / (UFC x TK x MF x V)
OEL = Occupational Exposure Limit
POD = Point of Departure for Extrapolation (mg/kg-day)
BW = Body Weight (kg)
UFC = Composite Uncertainty Factor
TK = Toxicokinetic adjustment
MF = Modifying Factor
V = Volume of air breathed during work shift (m3)
PERSONAL CARE PRODUCTS
Intimate health care
Baby care products
Engineered products/Medical devices
Over The Counter (OTC) products
“Any substance or mixture intended to be placed in contact with the external parts of the human body (epidermis, hair system, nails, lips and external genital organs) or with the teeth and the mucous membranes of the oral cavity with a view exclusively or mainly to ....”
Definition of a cosmetic
INDIA : 1940
Article intended to be rubbed, poured, sprinkled or sprayed on, or introduced into, or otherwise applied to, the human body or any part thereof for cleansing, beautifying, promoting attractiveness, or altering the appearance, and includes any article intended for use as a component of cosmetic.
→ Antibacterial hand washes not permitted as cosmetics in India but are cosmetics in EU. Feminine hygiene products are not cosmetics in India whereas it is a cosmetic in EU.
USA : 1936
“The term ‘cosmetic’ means articles intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to the human body or any part thereof for cleansing, beautifying, promoting attractiveness, or altering the appearance, and articles intended for use as a component of any such articles; except that such term shall not include soap”.
EU : 1976
“Any substance or preparation intended to be placed in contact with the various external parts of the human body (epidermis, hair system, nails, lips and external genital organs) or with the teeth and the mucous membranes of the oral cavity with a view exclusively or mainly to cleaning them, perfuming them, changing their appearance and/or correcting body odours and/or protecting them or keeping them in good condition.”
→ This means SPF products are “ cosmetics” in the EU and India but are OTC in USA.
toothpaste is not a cosmetic in US but is a cosmetic in India and EU
Cosmetic VS Drug
Fast moving consumer goods
Low biological / systemic activity
Inherently low risk
Slow moving goods
High biological / systemic activity
Inherently high risk
Composition of a Safety Assessment
The Safety Report for cosmetic products is composed of:
Part A –Safety information
Part B –Safety assessment
Part A: Cosmetic Product Safety Information
Part B: Cosmetic product Safety Assessment
The reasoning of the safety assessment
The related conclusion to the data in Part A has to be documented in detail.
Labelled warnings and instructions of use.
Description of the evaluation sequence,
Conclusion of the evaluation, justification for the conclusion.
Qualification of safety assessors.
Transfer of responsibility for part B (Signature).
Exposure Assessment - Cosmetic Ingredients
The following parameters describe an exposure scenario
Cosmetic product type (s) in which the ingredient may be used
Method of application as detailed as possible, e.g. rubbed-on, sprayed, applied and
washed off, etc.; considerations whether the product is a rinse-off or leave-on
product and which retention factor should be applied
Concentration of the ingredient in the marketed cosmetic product
Quantity of the product used at each application
Frequency of use
Total area of skin contact
Duration of exposure
Target consumer groups (e.g. children, people with sensitive, damaged or compromised .
skin) where specifically required
Application on skin areas exposed to sunlight
Location of use (indoors/outdoors) and ventilation
Phases In Risk Assessment of Personal Care Products (PCPs)
Exploratory Phase - Ingredient Hazard Assessment
Development Phase - Exposure Assessment & Ingredient Risk Assessment
Commercial & Market Phase - Product Safety Assessment & Post Market Compliance
Challenges In Risk Assessment of PCPs
Emerging safety concerns
Emerging safety issue
Rapidly gaining media attention
Global Move to eliminate EDs
Availability of new data
Updates in regulatory requirements
Ethical constraints in human testing
Pharmacokinetics is the study of the disposition of a drug.
The disposition of a drug includes the processes of ADME (Absorption, Distribution, Metabolism, Excretion).
Significance of knowing Pharmacokinetics
Patients may suffer:
Toxic drugs may accumulate
Useful drugs may have no benefit because doses are too small to establish
A drug can be rapidly metabolized.
Order of reactions
95% of the drugs in use at therapeutic concentrations are eliminated by first-order elimination kinetics and rest by Zero-order.
The Order of Reaction refers to the power dependence of the rate on the concentration of each reactant. Thus, for a first-order reaction, the rate is dependent on the concentration of a single species. A second-order reaction refers to one whose rate is dependent on the square of the concentration of a single reactant (e.g., in a homo-dimerization reaction, A + A → A2) or the combined first-order dependence on the concentrations of two different reactants (A + B → C).
Zero Order Reactions
Elimination of a constant quantity per time unit of the drug quantity present in the organism.
The rate of reaction is independent of the concentration of the reactants in these reactions.
A change in the concentration of the reactants has no effect on the speed of the reaction
Ethyl alcohol as a model of zero-order elimination kinetics
Ethanol particularly will be interesting to the intensivist, owing to the extreme fondness for it among the intensive care community. Rangno et al (1981) were able to plot some excellent zero-order elimination curves for IV-infused ethanol (a)and oral doses (b) among eight healthy male volunteers whom they lured into the experiment with the promise of booze. The high-dose group got fairly sozzled with a dose of 1.25g/kg, which for a 70kg male would be 87.5g (almost 9 standard drinks, more than a full bottle of wine), reaching a blood alcohol concentration of 0.2% - four times over the Australian legal driving limit.
Across all normal dose ranges, ethanol elimination appears to be quite linear, and independent of concentration. As seen from the Figure 1, the gradient of diminishing concentration over time from a high concentration is the same as that from a low concentration. This is because the dose consumed is typically quite high (i.e. it is a low potency drug). The enzymes responsible for its clearance in the normal person are saturated with the first drink; the Km of alcohol dehydrogenase for ethanol is about 1mmol/L (Cederbaum, 2012). The Vmax for alcohol ends up being about 7-10g/hr. It would, therefore, take an average person approximately 28 hours to completely metabolize one whole bottle of vodka, which explains some of the adverse functional effects seen during the subsequent morning.
Interestingly, the Km of alcohol dehydrogenase for other alcohols is much higher; the metabolism of methanol and ethylene glycol, therefore, proceed according to first-order kinetics. Of all the many isoforms of alcohol dehydrogenase, none show a preference for ethyl alcohol. It clearly has never been high on the list of metabolic priorities, as far as evolution is concerned.
A few substances are eliminated by zero-order elimination kinetics because their elimination process is saturated. Examples are Ethanol, Phenytoin, Salicylates, Cisplatin, Fluoxetine, Omeprazole.
First Order Reactions
Elimination of a constant fraction per time unit of the drug quantity present in the organism. The elimination is proportional to the drug concentration.
95% of the drugs in use at therapeutic concentrations are eliminated by first-order elimination kinetics.
The rates of these reactions depend on the concentration of only one reactant, i.e. the order of the reaction is 1.
In these reactions, there may be multiple reactants present, but only one reactant will be of first order concentration while the rest of the reactants would be of zero order concentration.
Example of a first order reaction: 2H2O2 → 2H2O + O2.
IMPURITY QUALIFICATION FOR PHARMACEUTICALS
Use of pharmaceuticals is always a balance of risks and benefits, the same is not true for impurities in pharmaceuticals; Impurities convey the only risk. Impurities should be contained below the level of concern or accepted general limits of thresholds in both drug substances and drug products. Impurities above threshold levels should be Toxicologically qualified.
Overview of the qualification process for :
Impurities in New Drug Substances
Impurities in New Drug Products
DNA Reactive (Mutagenic) Impurities In Pharmaceuticals to Limit Potential Carcinogenic Risk
WHAT IS THE IMPURITY?
Impurities are unwanted chemicals that remain with the active pharmaceutical ingredients (APIs), or develop during formulation preparation or upon aging of both API and formulated APIs to medicines.
Two types of impurities:
Drug substance impurities (starting materials, intermediates, degradation products)
Drug product impurities (degradation products, reaction products of the drug substance with excipients or the container-closure system)
Qualification is the process of acquiring and evaluating data that establishes the biological safety of an individual impurity or a given impurity profile at the level(s) specified.
ICH IMPURITIES GUIDELINES
Q3A(R2) : Impurities in New Drug Substances
(Current Step 4 version, dated 25 October 2006)
Q3B(R2) : Impurities in New Drug Products
(Current Step 4 version, dated 2 June 2006)
Q3C (R7) : Impurities: Guideline for Residual Solvents
(Current Step 4 version, parent guideline, 17/7/97, revised October 2018)
Q3D : Guideline For Elemental Impurities
(Current Step 4 Version, December 2014)
M7(R1) : Assessment And Control Of DNA Reactive (Mutagenic) Impurities In
Pharmaceuticals to Limit Potential Carcinogenic Risk
(Current Step 4 Version, March 2017)
ICH Q3A (R2): IMPURITIES IN NEW DRUG SUBSTANCES
Classification of Impurities
Impurities can be classified into the following categories:
Organic (process and drug-related)
Includes starting materials
Reagents, ligands, and catalysts
Inorganic (process related - known)
Ligands and catalysts
Heavy metals or other residual metals
Other materials (eg. filter aids, charcoal, etc.)
The organic or inorganic liquid used as a vehicle in preparation of Solution or
suspension during the synthesis of Drug substance
ICHQ3A indicates that sponsors should summarize the actual and potential impurities most likely arise during the synthesis, purification, and storage of the new drug substance.
ICH Q3B(R2): IMPURITIES IN NEW DRUG PRODUCTS
Identification, Reporting, and Qualification of Impurities
ICHQ3B(R2) addresses impurities in new drug products that are degradation products
of the drug substance resulted as a reaction product of the drug substance with an
excipient and/or immediate container closure system.
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